Nonmuscle myosin 2A (NM2A) not only plays numerous and diverse roles in vertebrate development, but also functions as a negative regulator for the formation of squamous cell carcinoma (SSC). Our previous research reported that ablation of NM 2A in mice (A-/A-) led to the abnormal formation of the visceral endoderm and embryonic lethality by E6.5. However, the molecular mechanisms responsible for A-/A- embryonic death were not fully characterized. Since in vitro differentiation of embryonic stem cells (ESCs) has been shown to faithfully mimic early embryonic development in vivo, we first established a mouse ESC differentiation system, including a spontaneous differentiation system (embryonic body formation) and a hematopoietic differentiation system (embryonic body formation with enhanced hematopoietic differentiation). Under spontaneous differentiation, a gene ontology analysis of down-regulated genes according to biological processes showed that NM 2A-/- ESCs has a developmental deficiency related to ear development, immune system development, vasculature development, hematopoietic or lymphoid organ development, and embryo implantation. RT-qPCR analysis revealed that NM 2A deletion decreased mesendoderm gene expression (Mesp1, Mixl1, T gene and Eomes). In addition, compared to four week old control teratomas, NM 2A knockout teratomas exhibited a low level of endoderm genes, such as Sox17, HNF4a, and AFP. Gene ontology analyses also showed that the top two downregulated biological processes in NM 2A-/A- ESCs are chromatin organization and chromatin modification. Compared to wild type, the levels of tri-methylation of K4, K9 and K27 on histone H3 were significantly decreased during differentiation. These data indicate that there is a difference in chromatin structures between the wild-type and the A-/A- ESCs in development. Defects in hematopoietic development are further confirmed in a hematopoietic differentiation system where NM 2A deletion decreased the percentage of hemangioblast cells (Flk1+ VE-Cad-) and hematopoietic progenitors (CD41+ CD45-). Interestingly, an inhibitor of the lysine demethylase 5 family (CPI-455) could rescue the defects in hematopoietic development caused by NM 2A deletion. The KDM 5 family contains 4 members including KDM 5A, KDM 5B, KDM 5C and KDM 5D. Knock down of KDM 5A could rescue the hematopoietic development defects caused by NM 2A deletion. These data indicate that KDM5A-mediated histone demethylation is critically involved in hematopoietic development defects caused by NM 2A deletion. Overall, these data indicate that deletion of NM 2A results in major defects related to chromatin modification and organization that consequently affects ESC differentiation including hematopoietic or lymphoid organ development.